1. Field of the Invention
Historically, fungal infections, and specifically yeast infections, have been difficult to treat due to a lack of specificity and potency of available antifungal compounds. Similarly, in the food and beverage fermentation industry, there exists a continuing need for antifungal agents such as cytotoxic proteins to insure culture purity. Cytotoxic proteins in general may also be toxic to other organisms and hence useful as biopesticides, such as insecticides, nematicides, and/or herbicides.
The invention relates to novel cytotoxic proteins produced by the yeast Pichia inositovora. The cytotoxic proteins possess antifungal activity and may be used for the treatment of fungal infections, or the prevention or control of fungal growth. The proteins are also potentially valuable as biopesticides.
2. Description of the Prior Art
Numerous filamentous fungi and yeasts have been discovered to contain linear double-stranded DNA plasmids [Meinhardt et al., Curr. Genet., 17:89-95 (1990), and Samac and Leong, Mol. Plant-Microbe Interac., 2:155-159 (1989)]. Among the yeasts that contain these elements are Saccharomyces kluyveri [Kitada and Hishinuma, Mol. Gen. Genet., 206:377-381 (1987)], Kluyveromyces lactis [Stark et al., Yeast, 6:1-29 (1990)], Phaffia rhodozyma [Wilber and Proffitt, Yeast Genetics and Molecular Biology Meeting, Jun. 16-21, 1987, Genetics Society of America, San Francisco, CA, Abstract 106], Saccharomycopsis crataegensis [Sheperd et al., Curr. Genet., 12:297-304 (1987)], and Pichia inositovora [Ligon et al., Plasmid, 21:185-194 (1989)].
Although most fungal linear plasmids are mitochondrial [Samac and Leong, ibid] in the yeasts K. lactis, S. cratagensis, and P. inositovora, these elements have been found to be located in the cytoplasm. Based on shared characteristics with other linear DNA plasmids, such as 5' ends protected by proteins and terminal inverted repeats, these cytoplasmic episomes have been included in a class of DNA molecules with the proposed name "invertrons" [Sakaguchi, Microbiol. Rev., 54:66-74 (1990)].
Functions have been ascribed to linear DNA plasmids of yeasts in the K. lactis system, consisting of two plasmids, pGKL1 and pGKL2, which are 8.1 and 13.4 kilobase pairs (kbp) in size, respectively. The smaller of the two plasmids, pGKL1, has been shown to encode both production of and immunity to a three subunit proteinaceous toxin active against a wide range of yeast species [Gunge et al., J. Bacteriol., 145:382-390 (1981)]. A function has not yet been discovered for the linear DNA plasmids of the yeasts S. kluyveri and S. crataegensis.